Small Molecule Lead Optimization to Increase Selectivity and Minimize Off-target Effects

Yu (Zoe) Zhong

Genentech, Safety Assessment, South San Francisco, CA

zhong.yu@gene.com

Conflict of Interest Statement

Yu (Zoe) Zhong is an employee at Genentech

Objectives/Outline• Objectives: To share the rationale and approaches on how to increase

selectivity and minimize off-target effects of small molecules – an important aspect of safety lead optimization

• Outline:• Why we care about off-target effects (secondary pharmacology) of small molecules

• What are the drivers of secondary pharmacology and How to minimize it

• How to contextualization the data• Case studies

• Conclusions

Two main sources of toxicity for small molecules

On-Target• aka ‘exaggerated pharmacology’

• Due to pharmacological engagement of the intended molecular target (primary pharmacology)

• Therapeutic index ~ 1

• Strategy – Target Safety Assessment: Does the potential on-target safety liability fit the indication (benefit/risk)?

Off-Target• Due to pharmacological engagement of unintended molecular target(s); and/or

other non-pharmacological toxicity (e.g. membrane damage)

• Physicochemical characteristics-driven

• ADME-related

• Strategy – Minimizing

Understanding secondary pharmacology is required by ICH S7A

• Secondary pharmacology –

‘Studies on the mode of action and/or effects of a substance not related to its desired therapeutic target’• Near targets

• Off-target pharmacological effects

• Data are included in regulatory filing • e.g. IND 2.6.2 Pharmacology section

Promiscuity is associated with greater toxicity and attrition

• Marketed Drugs: 5% promiscuous & 66% selective

• Withdrawn or Discontinued Drugs: 20-24% promiscuous; 40-50% selective

Bowes, et al. 2012. Nature Drug Discovery. 11:909

In Vitro Pharmacology Receptor Screens

Underlying assumption:

Compounds that bind numerous, unintended targets are associated with greater toxicity

Promiscuity index: High: >20% targets with >50% inhibitionMedium: 5-20% targets with >50% inhibitionLow: <5% targets with >50% inhibition

Physicochemical properties are fixed for each molecule and include the structural features and physical attributes

• Examples of physical attributes: molecular weight, lipophilicity(cLogP), acid-ionization constant (pKa), solubility, boiling & melting points, etc.

• Examples of structural features include substructures like thiophenes, anilines, basic amines, & acids, which determine the chemical interactions of a drug

thiophene Primary amine aniline Carboxylic acid

Physicochemical properties are the main drivers of off-target toxicity through multiple mechanisms

Lipophilic drugs

Basic amines

Reactive substructures

Acids

Promiscuity (non-selectivity), e.g. cardiovascular & NeuroIn Vivo Toxicity (tissue accumulation)

PromiscuityIn Vivo Toxicity (accumulation in tissues and acid organelles, e.g. phospholipidosis)

Genetic ToxicityPhototoxicityIn Vivo Toxicity (interaction with macromolecules and cellular stress)

Transporter substrates and/or inhibitors (hepatic; renal)Inhibition of enzymes (hepatic/mitochondrial)

• We do not always understand the mechanism of toxicity; however, known mechanisms are evaluated for all small molecules

Donna Dambach

Promiscuity

hERG inhibition

Phospholipidosis*

Rodent toxicity

High lipophilicity is a risk factor for promiscuity and in vivo toxicity

Challenges of low lipophilicity:PermeabilityRenal clearance

Solubility

Hepatic clearance

Challenges of high lipophilicity: A risk factor for solubility, metabolic stability, promiscuity (including hERG), tissue binding, in vivo toxicity

*enhanced for basic compounds ( pKa)

Rodent toxicity

Waring, et al. 2010. Expert Opin Drug Discov. 5:235

Increased lipophilicity is associated with toxicity in vivo

Hughes et al, 2008. Bioorganic Med Chem Lett 18: 4872-5.

• Compounds with low-ClogP/high-TPSA: ~2.5 times more likely to be clean as to be toxic

• High-ClogP/low-TPSA compounds: ~2.5 times more likely to be toxic as to be clean, representing an odds ratio of greater than 6

Strategy:

1)Work with your chemists to optimize physchem properties; in general decrease lipophilicity, and basicity

2)Utilize receptor panels to understand the promiscuity

*In vivo rat tolerability study (>=4 days); Toxicity assessed at a specific exposure threshold (10 uM Cmax total drug)

TPSA - total polar surface area

Secondary pharmacology profiling strategy

• Off target pharmacology can be observed/measured in a variety of systems

• In vitro recombinant/native cell lines (binding/functional)

• In vitro / Ex vivo tissue bath studies

• In vivo animal studies

• Panels• General panels with selected targets important for CNS, CV, GI safety (kinases, G protein-coupled

receptors, ion channels, transporters, nuclear receptors and enzymes)

• Target-specific panels to examine near targets (e.g., kinase panel, protease panel, ion channel panels for respective primary targets in that class)

• Adjust based on primary target, indication, chemical space, company experience

Initial screen

Tissue/in vivo translation for contextualization of the in vitro finding

Example: General secondary pharmacology panel

Bowel et al. (2012) Nat Rev Drug Discov. 11: 909.

Alignment of secondary pharmacology profiling to the drug discovery and development process

Bowel et al. (2012) Nat Rev Drug Discov. 11: 909.

• Early lead identification: Hazard identification of initial lead series; influence SAR, and compound optimization

• Candidate selection: Risk assessment – functional follow up, safety margin estimation • Sufficient safety margin to hCmax, free at efficacious exposure?

• Investigation: Contextualization of in vivo non-clinical / clinical findings • Can in vivo findings be explained by coverage of the off-target?

Interpretation and contextualization of binding hits

• Promiscuity:• Measured as: ratio (%) of targets with ≥50% binding

inhibition over total N of assays at 10 μM

• A measure of propensity of a molecule to bind other targets

• Promiscuity in a small panel is a surrogate for promiscuity across proteins in the body

• Selectivity:• Measured as: targets with ≥75% binding inhibition

in radioligand binding assays

• Evaluate each target “hit” for potential functional translation and in vivo implication

Promiscuity index: High: >20% targets with >50% inhibitionMedium: 5-20% targets with >50% inhibitionLow: <5% targets with >50% inhibition

Follow-up for binding hits: Determining functional translation and in vivo relevance

Binding Assay

• Direct measure of affinity

• Single, defined site of binding

only on the target

• No differentiation of modes of

action

• Multiple binding sites on one

target

• Functional Translation - end

result of binding at any site

on the target

• Agonist or Antagonist

ER

receptor

IP3

emission

Fluo4 NW

Ca2+

Gq

PLC

Functional AssayIn vivo

Assumptions:

• Free Drug Hypothesis: only free

(unbound) drug is available to

interact with the target

• Free Cmax frequently used as a

relevant (and more conservative)

measure of drug exposure

Dolo Diaz

Follow-up for binding hits: Determining functional translation and in vivo relevance

Binding Assay

• Direct measure of affinity

• Single, defined site of binding

only on the target

• No differentiation of modes of

action

• Multiple binding sites on one

target

• Functional Translation - end

result of binding at any site

on the target

• Agonist or Antagonist

ER

receptor

IP3

emission

Fluo4 NW

Ca2+

Gq

PLC

Functional AssayIn vivo

Assumptions:

• Free Drug Hypothesis: only free

(unbound) drug is available to

interact with the target

• Free Cmax frequently used as a

relevant (and more conservative)

measure of drug exposure

Dolo Diaz

Decision making:• How many off-targets are

affected?• Which off-targets are

affected?• What is the safety margin?

Case study: Syncope observed in dogs with a small molecule -consistent with alpha adrenergic receptor blockade

* Binding does not discriminate agonists vs antagonists; ** nonspecific subtypes that do not indicate subtypes

In Life Observations (PK study GNE#1 at 1 mg/kg intravenously in dogs): • Hypoactive at 2 min post dose and could not stand up (4 min post dose) • Heart rates doubled - 115-121 bpm (baseline) to 200-208 bpm (~12 min post dose)• Heart rate and normal activity recovered by ~ 45 - 58 min post dose

Eric Harstad

Case study: Syncope observed in dogs with a small molecule -consistent with alpha adrenergic receptor blockade

• Tachycardia is a normal physiologic response to hypotension in attempt to maintain blood pressure (BP)

– Alpha adrenergic receptors maintain normal BP

• GNE#1 potently antagonizes alpha 1a receptors (IC50 = 86 nM)

• Unbound plasma GNE#1 at C0 (1.2 uM) is ~14x higher than IC50

• Recovery could be based on cleared drug or compensation to raise BP

• GNE#2 was tolerated in dogs without similar clinical effects

• GNE#2 is ~10x less potent at the alpha 1a receptor and

• Unbound plasma GNE#2 at C0 ~40 nM is 21x below the alpha 1a IC50.

• Weaker inhibition observed at alpha 1b, but not other alpha receptors

* Binding does not discriminate agonists vs antagonists; ** nonspecific subtypes that do not indicate subtypes

In Life Observations (PK study GNE#1 at 1 mg/kg intravenously in dogs): • Hypoactive at 2 min post dose and could not stand up (4 min post dose) • Heart rates doubled - 115-121 bpm (baseline) to 200-208 bpm (~12 min post dose)• Heart rate and normal activity recovered by ~ 45 - 58 min post dose

Receptor Binding Data* (% displacement of ligand

at 10 uM)

GNE#1 GNE#2

alpha1** 82 75

alpha2** 62 39

Functional Assay - IC50 (nM)

Receptor GNE#1 GNE#2

alpha1a 86 840

alpha1b 4700 920

alpha2a 32000 37000

alpha2b 29000 15000

alpha2c - 71000

Eric Harstad

Selectivity on near targets important for safety margin considerations

• Target-specific

• Between primary pharmacological target and its homologues/isoforms

• Need to be considered early in project so assays can be in place for proactive lead optimization

• In vivo therapeutic index maybe smaller than in vitro selectivity, e.g. • IC75 or IC90 for efficacy vs. IC50 or IC20 for toxicity

• Ctrough needed for efficacy vs. Cmax-driven toxicity

• Assess off-target risks in the context of expected in vivo exposures (free) to understand potential therapeutic index

Conclusions

• Off-target pharmacology (secondary pharmacology) and promiscuity can be a significant safety attrition source

• Strategies to minimize promiscuity and enhance selectivity can be achieved by

• Optimize the physicochemical properties of a molecule, avoid known structural alerts

• Measure secondary pharmacology on near targets and unrelated targets

• In vitro ligand binding and cell-based function assays for hazard identification, and risk assessment

• Pay special attention to near targets where selectivity might be challenging

• Contextualize in vitro selectivity data with in vivo findings, taking into account unbound exposure at efficacious dose range

Reference

• Bowel et al. 2012. Nat Rev Drug Discov. 11: 909

• Hughes et al. 2008. Bioorganic Med Chem Lett. 18: 4872

• Waring et al. 2010. Expert Opin Drug Discov. 5: 235

Acknowledgement

• Dolo Diaz

• Donna Dambach

• William Proctor

• Eric Harstad

• Satoko Kiyota

Abbreviations

• ICH - The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use

• IND - investigational new drug application• LLE - ligand-lipophilicity efficiency• CNS - central nervous system• CV - cardiovascular • GI - gastrointestinal• SAR - structure activity relationship• Cmax - maximum (or peak) serum concentration of a drug• Ctrough - lowest concentration of a drug• hERG - the human Ether-à-go-go-Related Gene, codes Kv11.1, the alpha subunit of a potassium ion channel• cLogP - Calculated logP value of a compound, which is the logarithm of its partition coefficient between n-

octanol and water log(coctanol/cwater), a well established measure of the compound's hydrophilicity• TPSA - topological polar surface area of a molecule• BP - blood pressure• IC50 - the concentration of an inhibitor that corresponding to 50% of maximum inhibition effect